TAT Protein for preventing or treating AIDS

ABSTRACT

The invention relates to a method of preventing or treating acquired immunodeficiency syndrome (AIDS) in a patient, wherein the patient is administered with a Tat protein comprising amino acid sequence SEQ ID NO: 1 or 2, or a variant thereof capable of stimulating an immune response against Tat proteins.

The present invention provides compositions and methods for preventingor treating acquired immunodeficiency syndrome (AIDS) in a humanpatient, and a vaccine against an HIV-infection.

TECHNICAL BACKGROUND

The search for a vaccine against AIDS remains a major issue since thediscovery of the HIV-1 (Barre-Sinoussi et al., 1983). The failure ofclassical vaccine approaches targeting the HIV-1 envelop proteins pointsout the interest to use another target such as the HIV-1 trans-activatorof transcription protein called Tat, due to its extra cellular functioninvolving the collapse of the immune cellular response against HIVinfected cells (Jeang et al., 1999).

Tat exists predominantly in two different lengths, 86-87 residues or99-101 residues, that display multifaceted activities (Jeang et al.,1999). The long forms are predominant in clinical isolates from allHIV-1 subtypes except subtype D, due to the presence of a non-synonymoussingle nucleotide polymorphism, creating a stop codon in the second exonencoding sequence (Jeang et al., 1999). Tat is divided into six regions(Kuppuswamy et al., 1989) with one called basic region being involved inmost of the Tat activities. NMR studies of biologically active Tatvariants revealed that the basic region and the other functional regionsare well exposed to solvent and surround a core composed of part of theN-terminus with the well conserved Trp 11 (Péloponèse et al., 2000;Gregoire et al., 2001, Watkins et al., 2008). Among different Tatvariants this folding is similar in aqueous solution but can changedramatically when exposed to hydrophobic solvent (Péloponèse et al.,1999). Tat is a flexible protein and structural changes are necessaryfor it to bind to its pharmacological targets (Loret et al., 1992).

Tat is found mainly in the nucleus of infected cells where it functionsas a trans-acting transcriptional activator (Wong-Staal et al, 1985;Fujisawa et al., 1985), where it is known to be involved in theinitiation of transcription and RNA chain elongation (Cullen, 1990) by acomplex process involving interactions with cellular proteins and astem-bulge loop leader RNA, TAR (trans-activation responsive region), onthe viral mRNA and acetylation of the Tat (Bres et al., 2002). Studieshave shown that it also participates with the reverse transcription ofHIV-1 RNA (Harrich et al., 1997).

Despite the lack of a signal sequence, Tat is the only HIV-1 protein tobe secreted by HIV-1-infected cells and is found in detectable levels inthe culture supernatants of HIV-1-infected cells (0.1-1 ng/ml) (Ensoliet al., 1990; Westendorp et al., 1995b; Chang et al, 1997) and in thesera of HIV-1-infected patients (1-40 ng/ml) (Xiao et al., 2000;Westendorp et al., 1995b). Extracellular Tat display multifacetedactivities (Jeang et al., 1999), but the most important is to triggerapoptosis of uninfected T cells by traversing the cell membrane, leadingto apoptosis through the mitochondrial pathway (Chen et al., 2002,Campbell et al., 2004, de Mareuil et al., 2005). Neutralization ofextracellular Tat in vivo in SHIV-1 challenged macaques induces a riseof CD8⁺ T cells and HIV-1 infected CD4⁺ T cells become undetectable(Watkins et al., 2006).

Thus, the role of Tat in HIV-1 pathogenesis is not only as an essentialprotein for HIV-1 replication in infected cells, but also as anextracellular toxin (Gallo, 1999). Therefore, it is relevant to developa vaccine targeting Tat (Goldstein, 1996). However, seropositivepatients that have antibodies against Tat are unable to recognize Tatvariants from all HIV-1 subtypes (Campbell et al., 2007b). Moreover,these antibodies fail to slow disease progression to AIDS (Senkaali etal., 2008).

It is clear that a Tat vaccine using a B subtype Tat found mainly inEurope and North America as previously proposed (Godstein, 1996, Zaguryet al., 1998, Cafaro et al., 1999) has a low probability to provide atherapeutic and a preventive effect against HIV-1 infection in the restof the world and particularly in Africa. Moreover, a Tat vaccine using aB subtype Tat has also a low probability to provide a therapeutic and apreventive effect even in Europe and North America due to the incapacityof the immune system to neutralize extra cellular Tat.

The two main vaccine strategies against Tat up to now use a short, 86residue version of a B-subtype European Tat variant that is eitherinactivated (Zagury et al., 1998) or has full activity (Cafaro et al.,1999). These two approaches were tested on macaques followed by ahomologous SHIV-1 challenge (Cafaro et al., 1999; Pauza et al., 2000). Asignificant decrease of viremia was observed in these two studiescarried out respectively on Cynomolgus (Cafaro et al., 1999) and Rhesusmacaques (Pauza et al., 2000), without showing complete protectionduring primo-infection. Another study showed a long term control ofinfection following SHIV-1 challenge on Tat vaccinated Cynomolgusmacaques (Maggiorella et al., 2004). These studies point out thatvaccines using biologically active Tat appears to be a safe approach asindicated by safety studies carried out on monkeys in which no local orsystemic toxicity or adverse effects were observed (Cafaro et al., 1999,2001; Caselli et al., 1999, Goldstein et al., 2000).

It is interesting to note that conflicting results appears in Tatvaccine studies on macaques since no protection was observed with a SIVchallenge (Allen et al., 2002) or a vaccination with a recombinant viruscoding for a Tat-Rev protein (Verrier et al., 2002). These conflictingresults could be explained by a different immunization regimens, viralstock, routes of viral challenge and animal species. The differencebetween SIV Tat and HIV-1 Tat in the first study and the probabilitythat a Tat-Rev recombinant protein does not have the native Tat foldingor the native Rev folding for the second study may explain the absenceof protection. More puzzling, however, is the result of two otherstudies using similar viral vectors expressing Tat, Env and Gag thatgive opposite conclusions. One study shows the efficacy of vectored Tat,but not Gag and Env (Stittelaar et al., 2002), while another studyshowed efficacy of vectored Gag and Env, but not Tat (Liang et al.,2005). The main difference in the two studies is that one is using ahomologous challenge using the Tat Bru sequence in both the vaccine andin the SHIV (Stittelaar et al., 2002) and the other a heterologouschallenge with the Tat Bru sequence in the vaccine and Tat Jr in theSHIV (Liang et al., 2005). HIV-1 Jr and HIV-1 Bru are B subtypes(FIG. 1) but their Tat sequences have non-conservative mutationsinducing conformational changes (Gregoire & Loret., 1996). Thesesmutations between the vaccine and the virus use for the challenge mightexplain the lack of efficacy of the Tat vectored vaccine in the secondstudy (Liang et al., 2005). It is clear that the second study moreclosely resembled reality since a vaccinated person will not likely beexposed to a homologous virus infection, but in that case why havehomologous Gag and Env been used (Liang et al., 2005)?

Over the last 20 years, HIV-1 vaccine studies that target the HIV-1envelope proteins have been tested using a homologous SHIV/macaque modeland have met with success (Feinberg et al., 2002). However, clinicaltrials were not successful (Vanichseni et al., 2004). This is due to thehigh genetic diversity of HIV-1 and this is why heterologous SHIVchallenge in macaques, with a genetically distinct virus, should be usedto determine if a vaccine can be effective against HIV-1 infection inhumans (Feinberg et al., 2002). If a successful homologous SHIVchallenge is helpful to show the interest of Tat vaccination in vivo,the development of a Tat vaccine in humans worldwide has to take inaccount the genetic diversity of Tat. It is important to note thatimmunization with the B subtype Tat Bru does not stimulate an efficientresponse against Tat variants from A and C HIV-1 subtypes thatcorresponds to 75% of the contamination in the world (Opi et al., 2002).

The interest to develop a Tat vaccine rose with the discovery thatseropositive long-term non-Progressor (LTNP) patients had a higher levelof Tat antibodies than seropositive Rapid Progressor (RP) patients (Reet al., 1995; van Baalen et al., 1997; Zagurry et al., 1998, Re et al.,2001; Addo et al., 2001, Belliard et al., 2003). It is interesting tonote that with a serum dilution of 1:1000, Tat Bru is recognized by only30% of the RP patients in Europe (Zagury et al., 1998) and only 10 to14% in Africa (Butto et al., 2003). This percentage can reach up to 50%in Africa if other Tat variants from subtypes A, C and D are tested(Campbell et al., 2007). This result outlines again how mutations in Tatvariants can affect immunogenicity but it shows also that a large amountof seropositive patients are unable to recognize Tat. Furthermore Tatantibodies in African RP patients have no effect on the progression toAIDS (Sankali et al., 2008). It shows that, for a majority of HIV-1infected patients, Tat is not recognized while this protein is presentin their blood and patients who recognize Tat cannot neutralize thisprotein. The reason why Tat is so badly recognized by the human immunesystem could be due to sequence similarity of the basic region of Tatwith epitopes found in human proteins such as the protamine. It is clearthat triggering an immune response against the Tat basic region (regionIV) would lead to autoimmune response on humans. Region IV is wellconserved among Tat variants (FIG. 1) but it is not recognized by serafrom HIV-1 infected patients (Campbell et al., 2007). It is interestingto note that two third of new born children from HIV-1 infected mothersucceed to escape to HIV-1 infection (Beattie et al., 2001). They areseropositive up to 18 months old and then retro seroconvert. This highproportion of new-born infants that resist to HIV infection excludesgenetic factors that could be due to an innate immunity against HIV. Itcould be possible that a repression of the immune system to recognizeTat may exist in adults but not among new-born infants since protamineappears with sexual maturation.

A better attention should be taken to natural immunity occurring inadults against HIV-1. Natural immunity against HIV-1 is observed in alow proportion of the human population and cover different mechanismsgoing from chemokine mutations to the capacity to produce neutralizingantibodies against HIV-1 envelope (see Marmor et al., 2006 for areview). Natural immunity can be innate or acquired, the latter being ofcourse the most interesting for a vaccine development. Patients withnatural immunity against HIV-1 can be exposed and persistentlyseronegative (EPS) or can be seropositive and long term non progressor(LTNP). In most cases this natural immunity turns out to be an innateimmunity. However there is a very rare category of patients highlyexposed to the virus that are resistant to HIV-1 due apparently to anacquired immunity. Kenyan sex workers who are EPS had been intenselystudied and their resistance to HIV-1 appears to be related to theircapacity to develop an efficient CD8 T cell response against HIV-1(McMichael & Rowland-Jones, 2001). However, the paradox is that the CD8T cell response in EPS Kenyan sew workers is five time lower inmagnitude than that of seropositive Kenyan sex workers who ultimatelydevelop AIDS (Alimonti et al., 2006). To make things even more puzzling,studies of similar cohorts of EPS patients in Ivory Cost, Vietnam andCambodia show that they have no HIV-1 specific CD8 T cell response but anatural killer (NK) cell responses (Scott-Algara et al., 2003; Jennes etal., 2006), antibodies against HIV-1 envelop proteins (Nguyen et al.,2006) or cellular factors affecting viral entry steps (Saez-Cirion etal., 2006). Although it was believed that these Kenyan EPS patients hadan innate immunity, it turned out that it was an acquired immunity atleast for some of them who became seropositive after a lapse in sex work(Kaul et al., 2001).

More interesting was the study of an EPS cohort identified in Gabon(Huet et al., 1989). During the eighties in Africa, it was observed in aremote area of Gabon called “Haut Ogooué” that HIV-1 infected patientswere not developing AIDS (Delaporte et al., 1988). An epidemiologicalsurvey was decided and carried out on 750 pregnant women for two yearsand 25 were identified as seropositive (Huet et al., 1989). From these25 seropositive women, 23 retro seroconverted and became EPS during thetwo years of the survey. Although EPS patients have normally nodetectable virus, it was possible to isolate and clone a HIV-1 strainfrom one patient called Oyi when she was still seropositive (Huet etal., 1989). Contrary to other EPS cohort of sex workers or drug usersthat were constituted many years after the first exposure to HIV, theGabon cohort was constituted during the primo infection and explains whyit was possible to clone a virus. All women infected with HIV-1 Oyi didretro seroconvert but maintained a CTL response against HIV-1 and hadantibodies against P24 (Huet et al., 1989). The high proportion of EPSphenotype in this cohort (92%) indicates that the retro seroconversionwas probably due to an acquired immunity and not an innate immunity. Tenyears after the publication of this epidemiological survey, the 23 womenwere in good health and the HIV-1 or a sign of infection was no longerdetectable in their blood. Furthermore HIV-1 infection appears to bevery low in Gabon compared to other central African countries (Delaporteet al., 1996). HIV-1 Oyi has genes similar to regular HIV-1 strainsexcept the tat gene, which had mutations never found in other Tatvariants (Gregoire & Loret, 1996). Immunization with Tat Oyi raisesantibodies in rabbits that are able to recognize different Tat variantseven with mutations of up to 38%, which is not possible with other Tatvariants (Opi et al., 2002). Tat Oyi appears to induce a humoral immuneresponse against a three-dimensional epitope that are conserved in Tatvariants and this humoral response could make possible to neutralizeextracellular Tat. The role of extracellular Tat was not known duringthe eighties and the presence of antibodies against Tat was not testedin this Gabon cohort (Huet et al., 1989).

Seven rhesus macaques were immunized with synthetic Tat Oyi complementedwith an adjuvant then a heterologous challenge with the European SHIVBX08 was carried out on Tat Oyi vaccinated macaques and controlmacaques. Tat Oyi vaccinated macaques had a lower viremia associatedwith a rise of the CD8 T cells compared with control macaques. Moreover,SHIV infected cells were no longer detectable at 8 weeks post-challengein Tat Oyi vaccinated macaques. It is interesting to note that themacaque that had the lowest viremia had no antibodies against SHIVenvelop proteins. The macaque was challenged again, made a short periodof seropositivity and seroconverted (Watkins et al., 2006). It wastherefore possible to reproduce experimentally on macaque what isobserved in the field with EPS patients. It is important to note thatthis “EPS macaque” had a detectable viremia (watkins et al., 2006). Thisis a very promising result because it was a heterologous SHIV challengeand it shows that it is possible to dramatically reduce the level of HIVinfected cells. This goal has never been achieved in seropositivepatients under HAART treatment.

SUMMARY OF THE INVENTION

The invention provides a method of preventing or treating acquiredimmunodeficiency syndrome (AIDS) in a patient, wherein the patient isadministered with a protein comprising amino acid sequenceWKHPGSQPKTACNNCYCKRCCLHCQVCFTKKGLGISYGRKKRRQRRRAPQDSKTHQVSLSKQPASQPRGDPTGPKES (SEQ ID NO:1), or a variant thereof capable ofstimulating an immune response against Tat proteins, or a polynucleotidethat encodes said protein or variant.

Preferably, the protein or variant is capable of stimulating an immuneresponse against 2, 3, 4 or more HIV-1 subtypes.

In a preferred embodiment, the protein or variant is capable ofstimulating an immune response against Tat proteins from two, three,four, five or more HIV-1 subtypes selected from A, B, C, D, E, F, G, H,J, and K.

Most preferably, the protein or variant is capable of stimulating animmune response against five HIV-1 subtypes, preferably A, B, C, D, andE.

Preferably, the protein comprises, or consists of amino acid sequenceMEPVDPRLEPWKHPGSQPKTACNNCYCKRCCLHCQVCFTKKGLGISYGRKKRRQRRRAPQDSKTHQVSLSKQPASQPRGDPTGPKESKKKVERETETDPED (SEQ ID NO:2).

SEQ ID NO:2 differs from HIV Oyi Tat protein described in W000/61067 bya cysteine at position 22, which restores transactivation ability. Theprotein used herein exhibits the functional activity of Tat, i.e. thatis capable of HIV-1 gene transactivation in a HeLa P4 cell assay (FIG.4).

Substituting serine by cysteine at position 22 strengthenedimmunogenicity of the original Tat Oyi sequence, as shown with sera froma cohort of 100 seropositive patients that recognize better Tat Oyi C22than Tat Oyi S22 (FIG. 2). The transactivation activity of Tat Oyi Cys22 confirms that the 3D structure is correct and allows to characterizethe protein for a clinical trial.

A further subject of the invention is a vaccine comprising as an activeingredient a transactivation competent Tat Oyi protein comprising aCysteine at position 22., preferably selected from SEQ ID NO:1 or SEQ IDNO: 2.

In a preferred embodiment, the vaccine comprises an adjuvant.

Still another subject of the invention is a method of immunizingwarm-blooded animals comprising injecting an effective amount of aprotein comprising at least one of SEQ ID NO:1 or SEQ ID NO: 2 and anadjuvant.

This method may further comprises the step of determining whether theprotein triggers an immune response against five or more HIV-1 subtypes,wherein the protein stimulates an immune response against Tat proteinsfrom at least five HIV-1 subtypes.

FIGURE LEGENDS

FIG. 1 is a graph showing detection of Anti-Tat IgG in three cohorts,one was Vietnamese EPS patients (n=25), the second was Frenchseropositive patients (n=100) and the third was French seronegativenaive patients (n=20). Three types of serological pattern were observedwith patients blood samples in the ELISA titration curves with Tatvariants representative of the five main HIV-1 subtypes. FIG. 1A showsthe recognition of six Tat variants obtained with a serum from aVietnamese EPS patient. The level of Tat antibodies is not the sameregarding the six Tat variants and Tat CM240 was the best recognized forthe Vietnamese patients. FIG. 1B shows another patient who recognizesonly one or two Tat variants and is mainly observed in the Frenchseropositive cohort (n=100). FIG. 1C is the absence of recognition ofany Tat variants (OD inferior to 0.1), and is typical of the Frenchseronegative cohort (n=20), but also half of the French seropositivecohort and a third of Vietnamese EPS cohort.

FIG. 2 is a graph showing recognition of seven Tat variants with sera ofthe French seropositive cohort (n=100). Tat variant representatives fromHIV-1 subtypes A (Ug11RP), B (HXB2), C (96BW), D (Eli) and E (CM240)correspond to the A, B, C, D and E letters. Letter O corresponds to TatOyi Ser22 and Oc22 corresponds to Tat Oyi Ser22. This experiment showsthat the Cys 22 mutation did not alter the recognition of Tat Oyi butrather strengthened immunogenicity of the original Tat Oyi sequence.This is due certainly to the cystein region that is more similar to Tatwild type.

FIG. 3 shows a mass spectroscopy of an aliquot from a purified subfraction having a single peak at 25 min in analytical HPLC. Themolecular weight (MW) observed corresponds almost exactly to the MWexpected of 11578 for Tat Oyi Cys 22. Purification was successful withno trace of peptides with a molecular weight inferior or superior to11578. The peak at 5784.5 corresponds to the shadow peak (mass dividedby two) of the peak at 11575.

FIG. 4 is a graph that shows a transactivation assay with HIV LTRTransfected HeLa Cells. The biological activity of the synthetic Tat OyiCys 22 was tested with a cellular culture assay using HeLa P4 cells witha bacterial lac-Z gene under the control of the HIV LTR. Active Tatprotein can cross cytoplasmic and cellular membranes and “transactivate”the lac Z gene as revealed by the cytoplasmic accumulation ofβ-galactosidase. 2×10⁵ cells/well were incubated in a 24-well plate at37° C., 5% CO2, in Dulbecco's modified Eagle's medium (DMEM)supplemented with 10% foetal calf serum. After 24 h, cells were washedwith PBS and Tat protein diluted in DMEM supplemented with 0.01%protamine (Sigma), and 0.1% bovine serum albumin (Sigma) was added. Oneconcentration of the two Tat proteins was tested (500 nM) in order toverify that the level of β-galactosidase is dose-dependent regardingextracellular Tat. After 16 h at 37° C., 5% CO2 cells were washed withPBS and lysed. Proteins were extracted, and the β-galactosidase contentwas measured with a commercial antigen capture enzyme-linkedimmunosorbent assay (β-galactosidase ELISA, Roche MolecularBiochemicals). Absorbance values were measured at 405 nm.

FIG. 5 shows Circular Dichroism (CD) spectra of Tat Oyi Ser 22 and TatOyi Cys 22. CD spectra were recorded at 20° C. in the presence of 20 mMphosphate buffer, pH 4.5, over a 260-178 nm range using a 100 μm pathlength on a JASCO Corp. J-810 spectropolarimeter (Japan). Data werecollected at 0.5 nm intervals using a step auto response procedure(JASCO). CD spectra are presented as Δε per amide. Protein concentrationwas 1 mg/ml for Tat Oyi Ser22 and 0.3 mg/ml for Tat Oyi Cys22. The twoCD spectra are identical and show that the cys22ser mutation does notinduce a change that is detectable by CD.

FIG. 6 shows molecular modeling of Tat Oyi Cys22. Molecular modeling wascarried out with the Insight II 2002 package including Biopolymer,Discover and Homology (Accelrys, San Diego, Calif.). Two models werebuilt corresponding to Tat Oyi Ser 22 (data not shown) and Tat Oyi Cys22. The models were obtained from Tat Eli NMR structure (Watkins et al.,2008) using the CV Force Field and gradient conjugate algorithm forenergy minimization. The two models were identical and the cys22sermutation does not induce a change that could be deduced from molecularmodeling. It is interesting to observe that the C-terminus containingthe Glu 100 mutation is very close of regions 3 and 4 that have the mostconserved sequences in Tat variants.

DETAILED DESCRIPTION OF THE INVENTION

The inventor observed that patients that resist to HIV-infection(Vietnamese EPS patients) are able to recognize the Tat variantsrepresentative of the five main HIV-1 subtypes (FIG. 1A). These resultsshow that Tat antibodies that have the capacity of cross recognitionregarding different Tat variants could restore an efficient cellularimmune response against HIV.

On this basis, the invention provides a method of preventing or treatingacquired immunodeficiency syndrome (AIDS) in a patient, wherein thepatient is administered with a protein comprising amino acid sequenceSEQ ID NO:1 or 2, or a variant thereof capable of stimulating an immuneresponse against Tat proteins.

Preferably the protein is capable of stimulating an immune responseagainst two, three, four or more HIV-1 subtypes. Most preferably theprotein is capable of stimulating an immune response against Tatvariants from at least five HIV-1 subtypes.

In a preferred embodiment, the patient is administered with a proteincomprising amino acid sequence SEQ ID NO:2, most preferably a proteinconsisting of amino acid sequence SEQ ID NO:2.

In another embodiment, the patient is administered with a proteincomprising a truncated form of sequence SEQ ID NO: 1 or 2. For instance,the protein may be truncated of 1 to 10 amino acids, preferably 1 to 8,preferably 1 to 6, preferably 1 to 4, preferably 1 or 2 amino acids atthe N-terminus. The patient may also administered with a proteincomprising amino acid sequence SEQ ID NO:1 or 2 truncated of 1 to 14amino acids, preferably 1 to 12, preferably 1 to 10, preferably 1 to 8,preferably 1 to 6, preferably 1 to 4, preferably 1 or 2 amino acids atthe C-terminus.

The protein to administer may be a variant deriving from SEQ ID NO:1 orSEQ ID NO:2.

Preferably, the protein variant differs from SEQ ID NO:1 or SEQ ID NO:2by conservative amino acid substitutions. Conservative amino acidsubstitutions are substitutions among amino acids of the same class.These classes include, for example, amino acids having uncharged polarside chains, such as asparagine, glutamine, serine, threonine, andtyrosine; amino acids having basic side chains, such as lysine,arginine, and histidine; amino acids having acidic side chains, such asaspartic acid and glutamic acid; and amino acids having nonpolar sidechains, such as glycine, alanine, valine, leucine, isoleucine, proline,phenylalanine, methionine, tryptophan, and cysteine.

Preferably the protein variant, capable of stimulating an immuneresponse against Tat proteins, preferably from at least five HIV-1subtypes, shows at least 80%, preferably at least 85%, preferably atleast 90%, preferably at least 95% identity with SEQ ID NO:1 or SEQ IDNO:2.

Homology or identity is measured using sequence analysis software suchas Sequence Analysis Software Package of the Genetics Computer Group,University of Wisconsin Biotechnology Center, 1710 University Avenue,Madison, Wis. 53705. Amino acid sequences are aligned to maximizeidentity. Gaps may be artificially introduced into the sequence toattain proper alignment. Once the optimal alignment has been set up, thedegree of homology or identity is established by recording all of thepositions in which the amino acids of both sequences are identical,relative to the total number of positions.

The protein variant to administer may also comprise chemicalmodifications, e.g. modification of peptide bonds into a pseudopeptidebond.

The term “pseudopeptide” refers to compounds that are similar to thereference peptide but in which one or more peptide bonds —CO—NH— aresubstituted by a bond that is equivalent to the peptide bond, called apseudopeptide, such as —CH2-NH—, —CH2-S—, —CH2-O—, —CO—CH2-CO—,—CH2-CH2- for example.

The term “variant” further encompasses substitution of one or severalamino acids by a chemical residue which is not a natural amino acid.Substitutions by L-amino acids is also included.

In another aspect of the invention, the protein is covalently bound to apolyethylene glycol (PEG) molecule by their C-terminal terminus or alysine residue, notably a PEG of 1500 or 4000 MW, for a decrease inurinary clearance and in therapeutic doses used and for an increase ofthe half-life in blood plasma. In yet another embodiment, the proteinhalf-life is increased by including the protein in a biodegradable andbiocompatible polymer material for drug delivery system formingmicrospheres. Polymers and copolymers are, for instance,poly(D,L-lactide-co-glycolide) (PLGA) (as illustrated in US2007/0184015,SoonKap Hahn et al).

In still a further embodiment, the protein is bound to, or is associatedwith, a carrier. Carriers are known in the art (Plotkin, 1999).Bacterial carriers (i.e., carriers derived from bacteria) include, butare not limited to, cholera toxin B subunit (CTB); diphtheria toxinmutant (CRM197); diphtheria toxoid; group B streptoccus alpha C protein;meningococcal outer membrane protein (OMPC); tetanus toxoid; outermembrane protein of nontypeable Haemophilus influenza (such as P6);recombinant class 3 porin (rPorB) of group B meningococci; heat-killedBrucella abortus; heat-killed Listeria monocytogenes; and Pseudomonasaeruginosa recombinant exoprotein A. Another carrier is keyhole limpethemocyanin (KLH). Examples of viral-derived carriers are known in theart and include hepatitis B surface antigen (HBsAg) particles andhepatitis B core antigen (HBcAg).

In a preferred embodiment, the protein variant, capable of stimulatingan immune response against Tat proteins, preferably from at least fiveHIV-1 subtypes, is capable of transactivation.

Advantageously the protein variant is capable of stimulating an immuneresponse against Tat proteins from HIV-1 subtypes A, B, C, D, and E (Esubtype HIV strains are observed mainly in South East Asia but are oftenconsidered as recombinant forms of A subtype strains).

The protein or variant may be a recombinant protein, or preferably achemically synthesized protein, that may be synthesized in a solid phasesynthesis and preferably fast FMOC strategy.

When the protein is produced by chemical synthesis, the polypeptideaccording to the invention may be synthesized in the form of a singlesequence, or in the form of several sequences which are then linked toone another. The chemical synthesis may be carried out in solid phase orin solution, these two synthesis techniques being well known to thoseskilled in the art. These techniques are in particular described byAtherton and Shepard in “Solid phase peptide synthesis (IRL pressOxford, 1989) and by Houbenweyl in “Methoden der organischen Chemie”[Methods in Organic Chemistry] published by E. Wunsch Vol. 15-I and II,Stuttgart, 1974, and also in the following articles, which are entirelyincorporated herein by way of reference: P E Dawson et al. (Science1994; 266(5186):776-9); G G Kochendoerfer et al. (1999; 3(6):665-71); etP E Dawson et al., Annu Rev. Biochem. 2000; 69:923-60.

The protein may also be produced using genetic engineering techniqueswell known to those skilled in the art. When the protein is produced bygenetic engineering, it comprises, at the NH 2-terminal end, anadditional methionine residue corresponding to the translation of thefirst initiation codon. These techniques are described in detail inMolecular Cloning: a molecular manual, by Maniatis et al., Cold SpringHarbor, 1989.

The protein may thus be obtained in purified form, i.e. in a formexhibiting a degree of purity of at least 80%, 85%, 90%, 95%, orgreater. The degree of purity is defined relative to the other proteinspresent in the mixture which are considered to be contaminants Thisdegree is evaluated by colorimetry of an SDS-PAGE using coomassie blue.Densitometric measurement of the bands makes it possible to quantify thedegree of purity. The degree of purity may also be measured byreverse-phase HPLC, by measuring the area of the various peaks.

Several types of therapeutic compositions can be used to elicit animmune response against Tat proteins from at least five HIV-1 subtypes.

A subject of the invention is a pharmaceutical composition comprising aprotein comprising amino acid sequence SEQ ID NO:1 or SEQ ID NO:2, or avariant thereof capable of stimulating an immune response against Tatproteins, preferably from at least five HIV-1 subtypes, in combinationwith a pharmaceutically acceptable carrier.

Immunogenic compositions, proposed to be suitable for use as a vaccine,may be prepared most readily directly from immunogenic Tat Oyi Cys 22proteins and/or peptides (e.g., SEQ ID NO.:1, 2, or fragments of SEQ IDNO.:1 or 2) prepared in a manner disclosed herein. Preferably, theantigenic material is extensively dialyzed to remove undesired smallmolecular weight molecules and/or lyophilized for more ready formulationinto a desired vehicle.

The preparation of vaccines which contain peptide sequences as activeingredients is generally well understood in the art, as exemplified byU.S. Pat. Nos. 4,608,251; 4,601,903; 4,599,231; 4,599,230; 4,596,792;and 4,578,770. Typically, such vaccines are prepared as injectables:either as liquid solutions or suspensions, solid forms suitable forsolution in, or suspension in, liquid prior to injection may also beprepared. The preparation may also be emulsified. The active immunogenicingredient is often mixed with excipients that are pharmaceuticallyacceptable and compatible with the active ingredient. Suitableexcipients are, for example, water, saline, dextrose, glycerol, ethanol,or the like and combinations thereof. In addition, if desired, thevaccine may contain minor amounts of auxiliary substances such aswetting or emulsifying agents, pH buffering agents, or adjuvants thatenhance the effectiveness of the vaccines.

Vaccines may be conventionally administered parenterally, by injection,for example, either subcutaneously or intramuscularly. Additionalformulations which are suitable for other modes of administrationinclude suppositories and, in some cases, oral formulations. Forsuppositories, traditional binders and carriers may include, forexample, polyalkalene glycols or triglycerides: such suppositories maybe formed from mixtures containing the active ingredient in the range of0.5% to 10%, preferably 1-2%. Oral formulations include such normallyemployed excipients as, for example, pharmaceutical grades of mannitol,lactose, starch, magnesium stearate, sodium saccharine, cellulose,magnesium carbonate and the like. These compositions take the form ofsolutions, suspensions, tablets, pills, capsules, sustained releaseformulations or powders and contain 10-95% of active ingredient,preferably 25-70%.

The proteins may be formulated into the vaccine as neutral or saltforms. Pharmaceutically acceptable salts, include acid addition salts(formed with the free amino groups of the peptide) and those which areformed with inorganic acids such as, for example, hydrochloric orphosphoric acids, or such organic acids as acetic, oxalic, tartaric,mandelic, and the like. Salts formed with the free carboxyl groups mayalso be derived from inorganic bases such as, for example, sodium,potassium, ammonium, calcium, or ferric hydroxides, and such organicbases as isopropylamine, trimethylamine, 2-ethylamino ethanol,histidine, procaine, and the like.

The vaccines are administered in a manner compatible with the dosageformulation, and in such amount as will be therapeutically effective andimmunogenic. The quantity to be administered depends on the subject tobe treated, including, e.g., the capacity of the individual's immunesystem to synthesize antibodies, and the degree of protection desired.Precise amounts of active ingredient required to be administered dependon the judgment of the practitioner. However, suitable dosage ranges areof the order of several hundred micrograms active ingredient pervaccination. Suitable regimes for initial administration and boostershots are also variable, but are typified by an initial administrationfollowed by subsequent inoculations or other administrations.

The manner of application may be varied widely. Any of the conventionalmethods for administration of a vaccine are applicable. These arebelieved to include oral application on a solid physiologicallyacceptable base or in a physiologically acceptable dispersion,parenterally, by injection or the like. The dosage of the vaccine willdepend on the route of administration and will vary according to thesize of the host.

In a preferred embodiment, the composition of the invention furthercomprises an adjuvant. The adjuvant may be selected from any substance,mixture, solute or composition facilitating or increasing theimmunogenicity of the protein which comprises amino acid sequence SEQ IDNO:1 or SEQ ID NO:2, or its variants, wherein the adjuvant is able toenhance an immune response. Examples of adjuvants include preferentiallycalcium phosphate but also ALUM phosphate, ALUM hydroxide, MontanideCpG, QS21, ISCOM or monophosphoryl lipid A.

Other methods of achieving adjuvant effect for the vaccine include useof agents such as aluminum hydroxide or phosphate (alum), commonly usedas 0.05 to 0.1 percent solution in phosphate buffered saline, admixturewith synthetic polymers of sugars (Carbopol) used as 0.25 percentsolution, aggregation of the protein in the vaccine by heat treatmentwith temperatures ranging between 70° to 101° C. for 30 second to 2minute periods respectively. Aggregation by reactivating with pepsintreated (Fab) antibodies to albumin, mixture with bacterial cells suchas C. parvum or endotoxins or lipopolysaccharide components ofgram-negative bacteria, emulsion in physiologically acceptable oilvehicles such as mannide mono-oleate (Aracel A) or emulsion with 20percent solution of a perfluorocarbon (Fluosol-DA) used as a blocksubstitute may also be employed.

In many instances, it will be desirable to have multiple administrationsof the vaccine, usually not exceeding six vaccinations, more usually notexceeding four vaccinations and preferably one or more, usually at leastabout three vaccinations. The vaccinations will normally be at from twoto twelve week intervals, more usually from three to five weekintervals. Periodic boosters at intervals of 1-5 years, usually threeyears, will be desirable to maintain protective levels of theantibodies. The course of the immunization may be followed by assays forantibodies for the antigens.

The protein composition according to the present invention may beprepared using any conventional method known to those skilled in theart. Conventionally, the protein is mixed with a pharmaceuticallyacceptable diluent or excipient, such as water or phosphate bufferedsaline solution. The excipient or diluent will be selected as a functionof the pharmaceutical form chosen, of the method and route ofadministration, and also of pharmaceutical practice. Suitable excipientsor diluents, and also the requirements in terms of pharmaceuticalformulation, are described in detail in Remington's PharmaceuticalSciences, which represents a reference work in this field.

Another pharmaceutical composition comprises a polynucleotide thatencodes amino acid sequence SEQ ID NO:1 or SEQ ID NO:2, or a variantthereof as defined above. Such pharmaceutical composition isadministered to a host, for instance injected (known as DNA vaccination)and said nucleic acid expresses in vivo the Tat polypeptide. Such DNAvaccines usually consist of plasmid vectors. The delivery of naked DNAhas shown to be poorly efficient, and some carriers are usually neededto improve the delivery and uptake of DNA into cells. Two types ofcarriers have been yet developed: (1) viral carriers (adenoviruses,lentiviruses, measles virus), or (2) non-viral carriers such as polymers(and especially cationic polymers), encapsulated-DNA (liposomes,comprising cationic lipids interact spontaneously and rapidly withpolyanions, such as DNA and RNA, resulting in liposome/nucleic acidcomplexes) or DNA linked to gold microparticles. Moreover, agents, whichassist in the cellular uptake of nucleic acid, such as calcium ions,bacterial proteins, viral proteins and other transfection facilitatingagents, may advantageously be used.

The compositions mentioned above may be administered via anyconventional route usually used in the field of vaccines, such as theparenteral (intravenous, intramuscular, subcutaneous, etc.) route. Inthe context of the present invention, intramuscular administration willpreferably be used for the injectable compositions. Such anadministration may advantageously take place in the thigh or armmuscles. The compositions according to the present invention may alsoadvantageously be administered orally. Administration via the nasal,vaginal or rectal mucosa may also be recommended in the context of thepresent invention. The administration may also be carried out by givinga single dose or repeated doses, for example on D0 and at 1 month, 3months, 6 months and 12 months. Injections at J0 and at 1 month and 3months, with a booster, the periodicity of which may easily bedetermined by the treating physician, will preferably be used.

Generally, each dose will comprise between about 5 μg to about 500 μg ofthe protein, preferably between 10 to 200 μg. A more preferred dosagemay be about 50 μg of the protein as immunogen. Other dosage ranges mayalso be contemplated by one of skill in the art. Initial doses may beoptionally followed by repeated boosts, where desirable.

The present invention is also intended to cover the pharmaceuticalcompositions for use as a medicinal product, in particular for use as aprophylactic or therapeutic vaccine against HIV infection.

According to a preferred aspect, a subject of the present invention isthe use of a protein as described herein, or a polynucleotide thatencodes said protein, for immunizing the human body. The presentinvention therefore preferably relates to a method for administeringsaid polypeptide or polynucleotide so as to induce a specific humoralresponse.

The present invention thus provides a method for inducing HIVneutralizing antibodies comprising administration of a quantity of apharmaceutical composition as defined above which is sufficient toinduce the said humoral response.

The expression “a specific humoral response” is intended to mean aresponse comprising the production of antibodies directed specificallyagainst a surface well conserved among Tat variants. The production ofspecific antibodies may be easily determined using conventionaltechniques well known to those skilled in the art, such as ELISA, RIA orwestern blot.

The Tat protein and variant used herein are candidates of value fordeveloping a vaccine which can be used for the protection and/ortreatment of a large number, or even all, of the individuals at riskfrom or infected with HIV.

A therapeutic effect is recognized if seropositive patients that have noantiviral treatment have a lower viremia, a rise of lymphocyte CD4 cellsand/or a lower content in lymphocyte CD8 cells.

More particularly, a therapeutic effect is recognized when 30% of acohort of at least 55 HIV seropositive patients maintain their viremiaunder 50 copies/ml after interruption of their HAART treatment for sixmonths.

In patients with declared AIDS, such anti-Tat vaccine could limit theincidence of certain pathological conditions such as Kaposi's sarcoma orneurological syndromes which appear to be linked to a direct action ofthe Tat protein following their secretion by HIV-infected cells.

In asymptomatic patients, the pharmaceutical composition of theinvention is expected to delay the progression toward AIDS due to alower viremia, a rise of lymphocyte CD4 cells and/or a lower content inlymphocyte CD8 cells. Such a vaccine would allow the patient to restorean efficient immune cellular response against HIV infected cells, asappears to be not the case at the onset of the infection. In particular,the restoration of the activity of the cytotoxic T lymphocytes (CTL), NKcells and macrophages, but whose activity is inhibited by Tat. Thevaccination would have the consequence of allowing the patients tobecome at least non progressors. It is herein provided a vaccination foreliminating HIV infected cells and providing a curative treatmentagainst AIDS.

The figures and examples illustrate the invention without limiting itsscope.

EXAMPLES

Materials and Methods

Protein Synthesis. The Tat proteins were assembled according to themethod of Barany and Merrifield (1980) on HMP preloaded resin (0.5-0.65mmol) (Perkin Elmer, Applied Biosystem Inc., Forster City, Calif.) on anautomated synthesiser (ABI 433A, Perkin Elmer, Applied Biosystem Inc.)

Transactivation Assay with HIV LTR Transfected HeLa Cells. Thebiological activity of the synthetic Tat Oyi Cer22 was tested with acellular culture assay using HeLa P4 cells with a bacterial lac-Z geneunder the control of the HIV LTR. Active Tat protein can crosscytoplasmic and cellular membranes and “transactivate” the lac Z gene asrevealed by the cytoplasmic accumulation of β-galactosidase. 2×10⁵cells/well were incubated in a 24-well plate at 37° C., 5% CO2, inDulbecco's modified Eagle's medium (DMEM) supplemented with 10% foetalcalf serum. After 24 h, cells were washed with PBS and Tat proteindiluted in DMEM supplemented with 0.01% protamine (Sigma), and 0.1%bovine serum albumin (Sigma) was added. One concentration of the two Tatproteins were tested (500 nM) in order to verify that the level ofβ-galactosidase is dose-dependent regarding extracellular Tat. After 16h at 37° C., 5% CO2 cells were washed with PBS and lysed. Proteins wereextracted, and the β-galactosidase content was measured with acommercial antigen capture enzyme-linked immunosorbent assay(β-galactosidase ELISA, Roche Molecular Biochemicals). Absorbance valueswere measured at 405 nm.

Circular Dichroism (CD) CD spectra were recorded at 20° C. in thepresence of 20 mM phosphate buffer, pH 4.5, over a 260-178 nm rangeusing a 100 μm path length on a JASCO Corp. J-810 spectropolarimeter(Japan). Data were collected at 0.5 nm intervals using a step autoresponse procedure (JASCO). CD spectra are presented as Δε per amide.

Molecular modeling: Molecular modeling was carried out with the InsightII 2002 package including Biopolymer, Discover and Homology (Accelrys,San Diego, Calif.). The models were obtained from Tat Eli NMR structure(Watkins et al., 2008) using the CV Force Field and gradient conjugatealgorithm for energy minimization.

Human sera. The exposed persistently seronegative (EPS) patients (n=25)were Vietnamese intravenous drug users (IDU) who had been highly exposedto the risk of HIV-1 infection through needle sharing and followed since1996 (Truong et al, 2003) and gave their agreement for scientificstudies (Tran et al, 2006). French human blood samples were obtainedfrom a group of 20 HIV-1 seronegative and 100 HIV-1 seropositiveCaucasian patients. They were collected in hospital centers inMarseille. Most of the HIV-1 seropositive patients are underantiretroviral therapy (HAART). Enrolled participants in the Frenchseronegative cohort were informed of the purpose of the study (inaccordance with the Helsinki Declaration of 1975 and revised in 1983).All blood samples were collected and frozen in the country of origin ofeach population.

ELISA assay. Antibodies against that in human or mice sera were detectedby standard Enzyme Linked Immunosorbent Assay (ELISA) using Maxisorp U96immunoplates (Nunc). Plates were coated for 16 h at 4° C. with 0.5μg/well for each Tat variants diluted in phosphate buffer 100 mM pH 4.5.Currently, there are five main HIV-1 subtypes in the world: subtypes Aand C are predominant (72%) and are found mainly in Africa, India andSouth America; subtype B (10%) is mainly found in Europe andNorth-America; subtype D is found mainly in Africa and subtype E (nowdescribed as the recombinant form CRF_(—)01AE) is found mainly in SouthEast Asia (Hemelaar et al., 2006). Tat variability follows thisgeographical diversity with mutations of up to 40% observed among Tatvariants from A, B, C, D and E HIV-1 subtypes, that do not modify Tatactivity but affect Tat immune properties (Opi et al., 2002, 2004). Thisis why we used Tat Ug 11 RP, Tat HXB2, Tat 92Br, Tat Eli and Tat CM240to represent HIV-1 subtypes A, B, C, D and E. These five syntheticproteins have the Tat biological activity and a size going from 99 to101 residues which is now the dominant form of Tat in the field (Jeanget al., 1999). The recognition of Tat Oyi Cys 22 and Tat Oyi Ser 22 wascompared on a same plate with these five Tat variants Plates were thenincubated for 1 h at RT with 200 μl of PBS supplemented with 2% milk.Sera (one serum for one plate) were added in different dilutions of PBSsupplemented with 0.2% milk and shacked gently for 1 h at RT. Secondaryantibody (anti-Human or anti mice IgG peroxidase from Sigma) diluted1:1000 in PBS supplemented with 0.2% milk was added to each well andshacked gently for 1 h at RT. Plates were washed 3× with 200 μl of 1×PBS/0.05% Tween 20 (Genaxis) between each subsequent step. Revelationwas carried out with 100 μl of ABTS(2,2′-azino-bis-3-ethylbenz-thiazoline-6-sulfonic acid from Sigma) addedto each well and absorbance was read in an EL800 universal microplatereader (BIO-TEK INSTRUMENT, INC) at 405 nm.

Example 1 Sera from Vietnamese EPS Patients can Recognize Tat Variantsfrom the Five Main HIV-1 Subtypes including Tat Oyi

Anti-Tat IgG detected in EPS patients: We were able to detect Tatantibodies in a cohort of EPS patients in Vietnam. We carried out ELISAtest with Tat variants representative of the five main HIV-1 subtypesincluding Tat Oyi on three cohorts, one was Vietnamese EPS patients(n=25), the second was French seropositive patients (n=100) and thethird was French seronegative naive patients (n=20). Three types ofserological pattern were observed in Elisa test (FIG. 1). The first oneis characterized by the specific recognition (OD superior to 0.1) of allTat variants and is observed only in the EPS cohort (n=25). FIG. 1Ashows the recognition of six Tat variants obtained with a plasma from aVietnamese EPS patient. The level of Tat antibodies is not the sameregarding the six Tat variants and Tat from HIV-1 CM240 (Can et al.,1996) is best recognized for this patient. The second serologicalpattern is characterized by the absence of specific recognition for atleast one Tat variant and is mainly observed in the seropositive cohort(n=100). FIG. 1B shows the recognition of only one Tat variant obtainedwith a serum from a French seropositive patient. Seropositive patientscan recognize more than one Tat variant but can not recognize all Tatvariants (Campbell et al., 2007). The third serological pattern is theno recognition of any Tat variants, which is characterized by an ODinferior to 0.1. FIG. 1C show no recognition of Tat variants obtainedwith a serum from a French seronegative patient. This serologicalpattern is observed in all volunteers of the French seronegative cohortsof naive patients, but also in half of the French seropositive patients,and in a third of the Vietnamese EPS cohort (data not shown). It isinteresting to note that Tat CM240 is the best recognized in theVietnamese cohort followed by Tat Oyi as shown in FIG. 1A. Tat CM240 isrepresentative of HIV-1 E subtype that is endemic in Vietnam (Lan etal., 2003). The good recognition of Tat Oyi in these Vietnamese patientsshows the specific immune characteristic of Tat Oyi.

No Cross Recognition of the Six Tat Variants Observed in SeropositivePatients:

The main difference with EPS patients is shown in FIG. 1B. Only half ofthe patients had Tat antibodies and their sera were generally able torecognize one or two Tat variants (FIG. 1B). This result is very similarto what was observed with a Uganda cohort of seropositive patients(Campbell et al., 2007). However the design of this study was differentsince five blood sampling was made for one year in the Frenchseropositive cohort. It was very interesting to note that antibodiesagainst Tat could disappear in some patients and when they have againantibodies against Tat, it is the same Tat variant that is recognized(data not published). Most of the French seropositive patients (75%)were infected by B subtype HIV-1 strain and 75% recognize effectivelyTat HXB2, Tat Oyi Ser 22 or Tat Oyi Cys 22. However it is interesting toobserve that Tat Oyi is better recognized than Tat HXB2, although theTat HXB2 sequence has the closest homology with European B-subtype Tatvariants. As for the EPS Vietnamese cohort, these results confirm thespecific immune characteristic of Tat Oyi.

We expected that the Ser/cys 22 mutation would not alter the immunerecognition of Tat Oyi. This was confirmed by the French seronegativecohort since the Cys 22 mutation did not alter the recognition of TatOyi. It is interesting to observe that the recognition of Tat Oyi Cys 22is even better compared to Tat Oyi S22 due probably to the homology ofthe cysteine rich region with wild type Tat variants.

Titration curves are often identical for Tat Ug 11RP and Tat CM240 inthe French seropositive cohorts (data not shown). This suggests that asimilar epitope is recognized in these two variants and increaseartificially the number of patients recognizing these two variants.Titration curves are different for Tat Ug 11RP and Tat CM240 inVietnamese EPS patients as illustrated in FIG. 1A where the top curve isdue to Tat CM240. This indicate that it exists another epitope common toTat CM240 and E subtype Tat variants in Vietnam. Titration curves arealso often identical between Tat Oyi and Tat HXB2 both for Vietnameseand French patients (data not shown).

FIG. 1 and FIG. 2 show that there is not a common epitope among Tatvariants since our Elisa test allows almost to identify the HIV-1subtype that infected the patients. Consequently, it makes veryimprobable the possibility that the detection of Tat IgG antibodies inEPS patients could be due to the infection from a pathogen that wouldnot be HIV-1.

Discussion

Tat Oyi (101 residues) has mutations never found in Tat variants frompatients that are rapid progressors (Gregoire & Loret, 1996), and weassume that these mutations give to Tat Oyi the capacity to have a 3Depitope corresponding to a well conserved region in Tat variants (Opi etal., 2002, Watkins et al., 2006). It is therefore surprising that TatOyi is very well recognized in the Vietnamese EPS cohort and thisrecognition cannot be explained by a common epitope with Tat HXB2.

This study reveals that Tat from HIV-1 CM240 identified in a Thaipatient (Carr et al., 1996) is the most recognized in Vietnamese EPSpatients. HIV-1 CM240 is typical of South-East Asia and this resultindicates that an acquired humoral response against Tat probablyoccurred in EPS and could be the cause of their resistance to HIV-1 inspite of multiple exposure. A specific humoral response was alsoobserved in this EPS Vietnamese cohort regarding CD4-GP120 complex(Lopalco et al., 2005). This EPS Vietnamese cohort has similar signs ofHIV-1 immunity regarding an EPS cohort of European patients (Lopalco etal., 2005), and the recognition of Tat CM240 confirms that the EPSVietnamese patients were HIV infected and successfully resisted to HIV-1infection. On the other hand, it appears that a third of the EPSpatients have no Tat antibodies detectable and their resistance to HIVis not due to Tat antibodies (data not shown) Innate immunity wasrecently identified in this very same Vietnamese EPS cohorts, where fivepatients had mutations in their CD4 lymphocytes cellular factorsaffecting HIV entry steps (Saez-Cirion et al., 2006). It is interestingto know that one of these patients is among the EPS patients that do notrecognize Tat in this study.

Tat antibodies have been detected in sera of seropositive patients and acorrelation was observed with the long term non progressor (LTNP) status(Zagury et al., 1998; Re et al., 2001; Belliard et al., 2003). DenaturedTat variants or peptides were used in these studies and Tat antibodieswas detected only in a third of the seropositive patients (Zagury etal., 1998). We developed a new protocol to detect Tat antibodies inusing full Tat variants at pH 4.5 to maintain the three dimensionalstructure of Tat and in starting the detection at low dilution (¼) ofblood samples. This new protocol makes possible to increase thedetection of specific Tat antibodies to 50% of seropositive patients. Itis however amazing that 50% of seropositive patients that have extracellular Tat in their blood do not recognize any Tat variants. In theother hand, although the concentration of Tat antibodies is twice higherin seropositive patients regarding EPS patient, the amount of Tatantibodies remains huge in EPS patients regarding the potential amountof HIV infected cells producing Tat. What is odd in the Vietnamese isthe recognition of Tat Oyi. In a first screening experiment with theVietnamese cohorts using a 1/1000 blood sample dilution, we were able todetect the presence of antibodies against Tat Oyi in 56% of the EPSpatients. The new protocol starting with ¼ blood sample dilution made itpossible to point out that other Tat variants were significantlyrecognized in the EPS cohort (FIG. 1A).

Conclusion:

Only half of the seropositive patients have antibodies against Tat andcannot recognized the six Tat variants. Moreover, these antibodiesagainst Tat can disappear. These characteristics might explain theirincapacity to neutralize extra cellular Tat. It was shown that Tat caninduce apoptosis in cytotoxic T lymphocytes (Westendorp et al., 1995)and inhibit macrophages due to the over expression of Fas ligand (Cohenet al., 1999). The only cellular response against HIV-1 in the EPScohort was due to natural killer (NK) cells and Tat can induce cellularapoptosis in NK cells (Poggi et al., 2002). Our results suggest that Tatantibodies that have the capacity of cross recognition regardingdifferent Tat variants could restore an efficient cellular immuneresponse against HIV. The presence of IgG recognizing specifically Tatamong a majority of patients in the EPS cohort suggests that an acquiredimmunity against Tat did occur and made possible a recovery of theircellular immunity. This acquired immunity could be due to mutations in aVietnamese Tat variant similar to mutations observed in Gabon with TatOyi. Among the mutations that are specific of Tat Oyi, Glu 100 appearsto be very interesting because the apparition of a negative charge couldinduce an immogenicity to a surface of Tat Oyi corresponding to a highlyconserved surface among Tat variants.

Example 2 Synthesis, Purification and Characterization of Tat Oyi Cys 22

Tat OyiCys22 (SEQ ID NO:2) was assembled in solid-phase peptidesynthesis. Before cleavage of the resin, 330 mg of crude material wereobtained from a theoretical amount of 707 mg. The synthesis yield was46.7% and is similar to the one observed with the synthesis Tat Oyi Ser22.

Resin and protecting groups were cleaved from the protein content withacid trifluroacetic. The protein content was first purified by aprecipitation with tertio buthyl Ether (TBE) to remove the freeprotecting groups that are soluble in TBE. The pullet was dried andre-suspended in a 0.1% TFA water buffer and then the resin was remove byfiltration (0.22 μm). The resin-free protein content (approximately 240mg) was freeze dried and re-suspended in 0.1% TFA water buffer atconcentration up to 20 mg/ml in four fractions of approximately 60 mgcalled F1, F2, F3 and F4. Each fraction containing around 15 OD units.

Purification was carried out on each fraction using a Beckmanhigh-pressure liquid chromatography (HPLC) apparatus with a Beckman C8reverse phase column (10×150 mm). Buffer A was water with 0.1% TFA andbuffer B was acetonitril (Merck) with 0.1% TFA. Gradient was buffer Bfrom 15-35% in 40 minutes with a 2 ml/min flow rate.

From the different sub-fractions obtained after C8 reverse phaseseparation, it was possible to detect the typical absorption spectrum ofTrp in sub-fractions. HPLC analysis was carried out with a Merck RP-8column (4.6×100 mm) with similar buffers but using a gradient from10-50% in 40 minutes with a 0.8 ml per minute flow rate. The subfractionhaving the typical absorption spectrum of Tip was characterized by asingle peak at 25 min that has the same retention time that Tat Oyi Ser22 (data not shown). The mutation did not change the hydrophobicity ofTat Oyi.

A mass corresponding to the expected 11578 MW of Tat Oyi Cys 22 wasobserved with the sub fraction that has a single peak at 25 min in HPLC(FIG. 3). No contaminant was observed by mass spectroscopy. It isinteresting to note that the same mass spectrum was obtained after sixmonth with Tat Oyi Cys22 stored at −22° C. The absence of degradation ofthe active principle is encouraging for a vaccination campaign.

The total amount of purified Tat Oyi Cys 22 corresponds to a third of ODunits at 280 nm and ⅕ of the total amount of protein content.

Further characterization such as transactivation assay (FIG. 4) andcircular dichroism (FIG. 5) was carried out with Tat Oyi Cys 22.Transactivation assays was carried out with Tat HXB2 as a control.Transactivation was observed with Tat Oyi Cys 22 with a level similar toTat HXB2, while the variant Tat Oyi Ser 22 is unable to transactivate.The Cys 22 mutation makes possible to have a biological test showingthat Tat Oyi Cys 22 has a structure similar to other active Tatvariants.

Circular dichroism (CD) was carried out on Tat Oyi Cys 22 and Tat OyiSer 22 the same day. CD spectra (FIG. 5) reveals that the mutationCys22Ser does not induce a structural change detectable with CD.

To evaluate a potential change that could be induced by the mutation butnot detected with CD, a molecular modeling of the Tat Oyi Cys22 and TatOyi Ser22 was carried out (FIG. 6). Molecular modeling confirms that themutation Ser22Cys does not induce a structural change. The onlydifference between Ser and Cys is a different atom (oxygen instead of asulfur) in the lateral chain. Sulfur and oxygen are on the same columnof the Mendelief table and share chemical similarity as nucleophil.Sulfur is bigger than oxygen and the hydrogen linked to sulfur is morelabile than the one linked to oxygen. That seems to be the onlyexplanation why Tat OyiCys22 is able to transactivate and not TatOyiSer22.

It is interesting to observe that the C-terminus containing the Glu 100mutation is very close of regions 3 and 4 that have the most conservedsequences in Tat variants.

Example 3 Immunization with Tat Oyi Cys 22

Immunization of Mice was carried out with Tat Oyi Cys 22 (SEQ ID NO:2).No toxicity was observed on mice with Tat Oyi Cys 22 concentration thatis 1000 fold higher compared to what is planned for clinical trials(data not shown).

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1. A method of preventing or treating acquired immunodeficiency syndrome(AIDS) in a patient, wherein the patient is administered with a proteincomprising amino acid sequence SEQ ID NO:1, or a variant thereof capableof stimulating an immune response against a Tat protein.
 2. The methodof claim 1, wherein the protein or variant is capable of stimulating animmune response against two, three, four or more HIV-1 subtypes.
 3. Themethod of claim 2, wherein the protein or variant is capable ofstimulating an immune response against five HIV-1 subtypes.
 4. Themethod of claim 1, wherein the patient is administered with a proteincomprising amino acid sequence SEQ ID NO:2.
 5. The method of claim 1,wherein the patient is administered with a protein consisting of aminoacid sequence SEQ ID NO:2.
 6. The method of claim 1, wherein the patientis administered with a protein comprising amino acid sequence SEQ IDNO:2 truncated of 1 to 10 amino acids, preferably 1 to 8, preferably 1to 6, preferably 1 to 4, preferably 1 or 2 amino acids at theN-terminus.
 7. The method of claim 1, wherein the patient isadministered with a protein comprising amino acid sequence SEQ ID NO:2truncated of 1 to 14 amino acids, preferably 1 to 12, preferably 1 to10, preferably 1 to 8, preferably 1 to 6, preferably 1 to 4, preferably1 or 2 amino acids at the C-terminus.
 8. The method of claim 1, whereinthe protein variant, capable of stimulating an immune response againstTat proteins from at least five HIV-1 subtypes, shows at least 80%,preferably at least 85%, preferably at least 90%, preferably at least95% identity with SEQ ID NO:1 or SEQ ID NO:2.
 9. The method of claim 8,wherein the protein variant differs from SEQ ID NO:1 or SEQ ID NO:2 byconservative amino acid substitutions.
 10. The method of claim 1,wherein the protein variant is capable of transactivation.
 11. Themethod of claim 1, wherein the protein or variant is capable ofstimulating an immune response against Tat proteins from two, three,four, five or more HIV-1 subtypes selected from A, B, C, D, E, F, G, H,J, and K.
 12. The method of claim 1, wherein the protein or variant is achemically synthesized protein.
 13. The method of claim 1, wherein theprotein or variant is a recombinant protein.
 14. A pharmaceuticalcomposition, comprising a protein comprising amino acid sequence SEQ IDNO:1 or SEQ ID NO:2, or a variant thereof capable of stimulating animmune response against Tat protein and capable of transactivation incombination with a pharmaceutically acceptable carrier.
 15. Thepharmaceutical composition of claim 14, wherein the protein or variantis capable of stimulating an immune response against 2, 3, 4 or moreHIV-1 subtypes.
 16. The pharmaceutical composition of claim 15, whereinthe protein or variant is capable of stimulating an immune responseagainst five HIV-1 subtypes.
 17. The pharmaceutical composition of claim14, for use as a prophylactic or therapeutic vaccine against a HIV-1infection.
 18. A method of preventing or treating acquiredimmunodeficiency syndrome (AIDS) in a patient, wherein the patient isadministered with a polynucleotide that encodes a protein comprisingamino acid sequence SEQ ID NO:1, or a variant thereof capable ofstimulating an immune response against Tat proteins, preferably from atleast five HIV-1 subtypes.
 19. A pharmaceutical composition, comprisinga polynucleotide that encodes protein comprising amino acid sequence SEQID NO:1 or SEQ ID NO:2, or a variant thereof capable of stimulating animmune response against Tat proteins, preferably from at least fiveHIV-1 subtypes, in combination with a pharmaceutically acceptablecarrier.
 20. The pharmaceutical composition of claim 19, for use as aprophylactic or therapeutic vaccine against a HIV-1 infection.
 21. Avaccine comprising as an active ingredient a transactivation competentTat Oyi protein comprising a Cysteine at position
 22. 22. The vaccine ofclaim 21, wherein the protein is selected from SEQ ID NO:1 or SEQ ID NO:2.
 23. The vaccine of claim 21, further comprising an adjuvant.
 24. Amethod of immunizing warm-blooded animals comprising injecting aneffective amount of a protein comprising at least one of SEQ ID NO:1 orSEQ ID NO: 2 and an adjuvant.
 25. The method of claim 24, furthercomprising the step of determining whether the protein triggers animmune response against five or more HIV-1 subtypes, wherein the proteinstimulates an immune response against Tat proteins, preferably from atleast five HIV-1 subtypes.